Development of a novel technique for axenic isolation and culture of thraustochytrids from New Zealand marine environments

Journal of Applied Microbiology ISSN 1364-5072 ORIGINAL ARTICLE Development of a novel technique for axenic isolation and culture of thraustochytrids from New Zealand marine environments S.L. Wilkens 1 and E.W. Maas 2 1 Marine Biodiversity and Biosecurity, NIWA, Kilbirnie, Wellington, New Zealand 2 Marine Biotechnology, NIWA, Kilbirnie, Wellington, New Zealand Keywords antibiotics, axenic isolation, biotechnology, microbiology, protist, thraustochytrid. Correspondence Serena L. Wilkens, Marine Biodiversity and Biosecurity, NIWA, Private Bag 14901, Kilbirnie, Wellington 6241, New Zealand. E-mail: s.wilkens@niwa.co.nz 2011 1788: received 18 October 2011, revised 18 October 2011 and accepted 9 November 2011. doi:10.1111/j.1365-2672.2011.05197.x Abstract Aims: To maintain axenic cultures of commercially important thraustochytrids, a novel procedure was developed for the isolation of zoospores and sporangium from heterotrophic seawater samples and axenic culture on solid media. Methods and Results: Thraustochytrid cultures were isolated from Whangapoua Harbour in North East New Zealand and subjected to two antibiotic and antifungal treatment regimes designed to eliminate bacteria and fungi. Antibiotic trial 1 was designed to determine the appropriate combination of antibiotics (including streptomycin penicillin, ampicillin, rifampicin, nalidixic acid, tetracycline, gentamicin and the antifungal agent nystatin). Antibiotic trial 2 determined the optimal dosing frequency and concentration of the antibiotics, and antifungal found to be the most promising in trial 1. Axenic cultures were then spread plated onto nutrient agar containing the optimal antibiotic cocktail, and pure thraustochytrid colonies were purified on solid media using standard microbiological techniques. Conclusions: Removal of bacteria and fungi was best accomplished using a mixture of three antibiotics and one antifungal; rifampicin (300 mg l )1 ), streptomycin penicillin (25 mg l )1 ) and nystatin (10 mg l )1 ) were incorporated in seawater samples and incorporated into cultures every 24 h for a minimum of 2 days. Significance and Impact of the Study: The axenic isolation and culture of marine thraustochytrids from a marine habitat in New Zealand have significant implications for the biotechnological development of these potentially valuable protists. This method has global significance as it is reasonable to assume it could be used throughout the world to obtain axenic thraustochytrid cultures. Introduction Thraustochytrids are single-celled marine heterotrophic protists which have been isolated from different habitats and substrates worldwide including coastal and offshore seawater, estuarine water, sediments, senescent macroalgae and fallen mangrove leaves (Lewis et al. 1999). They are commonly associated with organic matter in marine coastal and estuarine sandy environments (Bongiorini et al. 2004) and thought to play an important role in the degradation and remineralization of organic material (Bongiorini et al. 2004). By penetrating organic particles through an ectoplasmic network and injecting their degradative enzymes, thraustochytrids are able to break down complex organic substrates such as cellulose cell walls, thereby absorbing the nutrients they require (Bremer and Talbot 1995; Raghukumar et al. 1995; Raghukumar and Raghukumar 1999; Raghukumar 2002). Research has demonstrated extremely high densities of thraustochytrids in coastal waters and sediments, leading to the suggestion 346 Journal of Applied Microbiology 112, 346 352 ª 2011 The Society for Applied Microbiology

S.L. Wilkens and E.W. Maas Axenic isolation of marine thraustochytrids from NZ that these organisms play a far more important role in promoting carbon turnover than originally thought (Kimura et al. 1999; Santangelo et al. 2000; Bongiorini et al. 2004). Marine microbes such as thraustochytrids represent a diverse genetic resource and their potential for biotechnological applications is expanding. Thraustochytrids can contain high levels of polyunsaturated fatty acids (PUFAs), particularly docosahexaenoic acid, eicosapentaenoic acid and arachidonic acid (AA) (Raghukumar 2008). These are essential fatty acids (i.e. can only come from dietary sources) in the human diet and critical for the normal development of brain, eye and other tissues (Nagao et al. 2002; Nichols 2004; Nichols et al. 2004). Thus, there is now a high market demand for products such as dietary supplements, therapeutic drugs and aquaculture feeds that contain PUFA (Lewis et al. 1999). Thraustochytrids potentially represent one of the main sources of PUFAs in the microbial loop (Kimura et al. 1999). Given their potential as alternative sources of omega-3 fatty acids, antioxidants and enzymes (Bahnweg 1979; Bremer and Talbot 1995; Bongiorni et al. 2005), this group of marine microbes is currently at the forefront of biotechnological exploration. However, despite the seeming environmental and economic importance of these species, to date, few studies have reported on thraustochytrids from New Zealand environments, particularly techniques for axenic isolation and culture. Treatment with antibiotics is still the most common method for the removal of bacteria and other microbial contaminants from cultures, but it rarely results in a culture free from fungi or other contaminating microbes (Choi et al. 2002). Methods for obtaining algal axenic cultures have included growth on solid culture media (Guillard and Keller 1984), antibiotic treatment, sometimes in combination with other antibacterial agents (Hunt and Mandoli 1992; Cottrell and Suttle 1993), and repeated washing of cells with or without the addition of antibiotics (Hoshaw and Roswski 1973; Uribe and Espejo 2003). No single procedure or antibiotic is likely to provide an axenic culture, largely due to the wide variety of bacterial species present. Therefore, a single antibiotic is not necessarily going to remove the bacterial population (Maas et al. 2007). In addition, some antibiotics may have a deleterious effect on microbes (Divan and Schnoes 1982). Proving the axenic status of any culture may be problematical because of the varying requirements of the different bacterial species present and the fact that some may be nonculturable or intracellular (Gallacher and Smith 1999; Lu et al. 2000). The purpose of this study was to develop a quick, reliable procedure for the isolation of axenic thraustochytrid strains from marine environments which can then be used to study the biotechnological potentials of the strains. No published method currently exists for the axenic isolation and complete removal of bacterial and fungal contamination from thraustochytrid cultures. Materials and Methods Micro-organism collection Estuarine water, sediment and mangrove leaves were collected in sterile plastic bottles during May 2006 from Whangapoua Harbour, North East New Zealand (Fig. 1). Leaves and sediment were collected and placed into 150 ml sterile saline. All samples were dusted with sterilized pine pollen (Porter 1990) and refrigerated for transport to the laboratory. Water, sediment and leaf samples were then incubated at 17 C for 2 weeks, under a continuous light regime (5 lmol m )2 s )1 ). Once thraustochytrids had colonized pine pollen grains, 1 ml of subsamples was taken using a pipette and was used to inoculate 100 ml sterile seawater, which was then used as stock cultures for antibiotic trial 1. The same protocol was used to Firth of Thames Whangapoua Harbour Thames Figure 1 Location of Whangapoua Harbour, New Zealand. Journal of Applied Microbiology 112, 346 352 ª 2011 The Society for Applied Microbiology 347

Axenic isolation of marine thraustochytrids from NZ S.L. Wilkens and E.W. Maas establish stock cultures for antibiotic trial 2, 2 weeks later, from the same enrichment. Antibiotic trial 1 The effect of different antibiotics and antifungal agent on the bacterial assemblages associated with thraustochytrids was tested using three different agar previously used for thraustochytrid culture; Seawater complete (SWC) (Haygood and Nealson 1985), Modified Vishniacs agar (KMV) (Porter 1990) and By+ agar (Atlas and Parks 1997), and three combinations of antibiotic and antifungal agents (summarized in Table 1). The most successful antibiotic combination was then used in antibiotic trial 2. SWC comprised agar (Oxoid) 12 g, yeast extract (Oxoid) 3 g, peptone (Oxoid) 5 g, glycerol (Sigma) 3 ml, 70% artificial seawater (Aquarium Systems Instant Ocean, USA) 1000 ml. KMV comprised agar (Oxoid) 12 g, glucose (Oxoid) 1 g, gelatine hydrolysate (Sigma) 1 g, yeast extract (Oxoid) 0Æ1 g, peptone (Oxoid) 0Æ1 g, 70% artificial seawater (Aquarium Systems Instant Ocean) 1000 ml. By+ comprised agar (Oxoid) 12 g, glucose (Oxoid) 5 g, yeast extract (Oxoid) 1 g, peptone (Oxoid) 1 g, 70% artificial seawater (Aquarium Systems Instant Ocean) 1000 ml. These agars were chosen as they differ in carbohydrate, carbon and nitrogen concentration and thereby provide three ways of assessing different bacterial populations and fungal growth. Four nonaxenic stock cultures were prepared (as described in Micro-organism collection) for each of the antibiotic treatments (Table 1), and the antibiotics were added directly to the culture bottle (the fourth remained untreated as the control). A 1 ml of subsample of each treated stock culture was serially diluted using sterile seawater, and the 10 )1,10 )3 and 10 )5 dilutions were spread plated in duplicate. Prior to adding antibiotic treatments, a day-0 subsample was taken from each stock culture and directly spread plated on each agar Table 1 Antibiotic treatments used for trial 1 Antibiotic antifungal Stock concentration (mg ml )1 ) AB1 AB2 AB3 Streptomycin 5000 50 300 100 penicillin Ampicillin 100 20 Rifampicin 25 25 Na-Nalidixic Acid 50 5 30 Tetracycline 100 2 Gentamicin 50 120 Nystatin 8 10 10 10 Control AB1, antibiotic treatment regime 1; AB2, antibiotic treatment regime 2; AB3, antibiotic treatment regime 3. type, in duplicate. Serial dilutions and spread plating from antibiotic treatments were repeated on days 6 and 12. Agar plates were incubated for 3 days at 17 C before visual inspection, and bacterial colony forming units per millilitre were calculated for each culture. Presence of thraustochytrid colonies was also visually confirmed by light microscopy to ensure antibiotic treatment did not negatively affect cell growth (i.e. cell counts using a haemocytometer and slide smears). In addition to visual observations, a 20 ll of subsample was taken from each culture on day 12, smeared onto a slide, gram stained and observed under light microscopy to confirm the removal of bacteria and fungi from the thraustochytrid culture [as 95% of marine bacteria are deemed unculturable and would not form visible colonies on plates (Su et al. 2007)]. Antibiotic trial 2 The impact of a combination of specific antibiotics and an antifungal (as determined in trial 1) using two concentrations and serial dosing the seawater stock cultures with antibiotics every 24 h was used to assess bacterial growth. On day 0, preprepared nonaxenic stock cultures were dosed with one of the two antibiotic treatments (Table 2), and third untreated stock culture was the control. A 1 ml of subsample of each treated stock culture was serially diluted using sterile seawater, and the 10 )1,10 )3 and 10 )5 dilutions were spread plated in duplicate onto the three agar types described previously. Stock cultures were repeatedly treated with antibiotics every 24 h for 4 days and subsequent subsamples diluted and spread plated. Each agar plate was then incubated for 3 days at 17 C before visual inspection, and bacterial colony forming units per millilitre were calculated for each culture. A 20 ll of subsample was taken from each culture on day 4, smeared onto a slide, gram stained and observed under light microscopy to confirm the removal of bacteria and fungi from the thraustochytrids cultures. Presence of thraustochytrid colonies was visually confirmed by light microscopy to ensure antibiotic treatment did not negatively affect cell growth. Table 2 Antibiotic treatments used for trial 2 Antibiotic antifungal Stock concentration (mg ml )1 ) AB2 AB2-B Streptomycin penicillin 5000 300 500 Rifampicin 25 25 50 Nystatin 8 10 10 Control AB2, antibiotic treatment regime 2; AB2-B, antibiotic treatment regime 2, at higher concentration. 348 Journal of Applied Microbiology 112, 346 352 ª 2011 The Society for Applied Microbiology

S.L. Wilkens and E.W. Maas Axenic isolation of marine thraustochytrids from NZ Statistical analysis anova and Tukey post hoc tests were used to analyse bacterial count data. (a) Results Antibiotic trial 1 Bacterial counts obtained from the antibiotic-treated and control thraustochytrid cultures are shown in Fig. 2(a c) for each of the growth medium used. The broad spectrum of the antibacterial and antifungal agents used significantly reduced the bacterial populations in each of the treatments, compared with the control. On SWC agar, antibiotic treatments AB2 and AB3 significantly (P < 0Æ05) reduced the bacterial population by 2 3 log 10 compared with the control, although there was slight recovery on day 12 with AB3 (Fig. 2a). AB1 was not as successful at reducing the bacterial levels in the thraustochytrid stock culture, and only 1 log 10 reduction (P < 0Æ05) was observed between this treatment and the control. Therefore, there was an overall and significant reduction in bacterial numbers when the stock culture was treated with AB2. On KMV agar, AB3 was significantly more successful at reducing the bacterial numbers (Fig. 2b). By day 12, the bacterial numbers were reduced to 5Æ0 10 1 CFU ml )1 compared with 3Æ5 9Æ3 10 3 CFU ml )1 recorded for the control, AB1 and AB3. An initial decline in bacterial numbers was observed for AB2 during days 0 6; however by 12, an increase in bacterial number was recorded, with the cell count not significantly different to the control. The bacterial numbers observed for AB1 and the control by day 12 were not significantly different. On By+ agar, antibiotic treatments AB1 and AB3 were slower to produce a decrease in bacteria, although a trend towards overall reduction was observed (Fig. 2c). Treatment AB2 produced a faster decrease in bacterial numbers by day 6; however, this appeared to plateau through till day 12 and resulted in a significantly (P <0Æ05) lower bacterial count of 5Æ0 10 2 CFU ml )1 compared to the control. Bacterial numbers in the control cultures did not reduce significantly throughout the course of the trial, for any of the growth media. AB2 produced the largest decline in bacterial numbers in the first 6 days across all agar types, and therefore, this combination was further optimized in trial 2. No fungal cells were present when the slide smears were examined suggesting that 10 mg l )1 is a good concentration to eliminate contaminating fungi from these thraustochytrid cultures. CFU ml 1 0 2 4 6 8 10 12 Time (Days) Figure 2 Bacterial growth recorded on three agar types during antibiotic trial 1. (a) Seawater complete agar, (b) Modified Vishniacs agar, (c) By+ agar. CFU, colony forming unit. Solid line is the control culture, dashed line is treatment AB1, dotted line is AB2, and ( )is AB3. Error bars are equal to one standard deviation. (b) (c) Journal of Applied Microbiology 112, 346 352 ª 2011 The Society for Applied Microbiology 349

Axenic isolation of marine thraustochytrids from NZ S.L. Wilkens and E.W. Maas Trial 2 Bacterial numbers obtained from the two antibiotic-treated cultures and control thraustochytrid culture are shown in Fig. 3(a c) for each of the bacterial growth medium used. Both antibiotic concentrations resulted in significantly (P < 0Æ01) lower bacterial counts post-treatment compared to the control. On SWC (Fig. 3a), a single dose of AB2 and AB2-B to the thraustochytrid cultures reduced the bacterial numbers by approximately 0Æ5 log 10 CFU ml )1 ; however, a second dose of AB2-B reduced the population by approximately another 4 log 10 CFU ml )1. In contrast, four doses of AB2 were required to produce the same reduction in bacterial numbers. On KMV (Fig. 3b), two doses of both treatments resulted in a 4Æ5 log 10 CFU ml )1 reduction in bacterial numbers, compared to the untreated control culture. Similar overall reduction in bacterial numbers was observed for both antibiotic dosage regimes. On By+ (Fig. 3c), the rate of decline in bacterial numbers was approximately the same, but an extra treatment of AB2 was required to completely remove the bacteria from the thraustochytrid culture. The control cultures did not show any decline in bacterial numbers throughout the course of the trial, on any of the bacterial growth media. On all of the bacterial media, treatment of the thraustochytrid cultures with AB2-B resulted in complete removal of bacteria after 2 days. However, treatment with AB2 did result in a reduction, but over comparably longer timeframe (2 4 days). The rate of bacterial reduction was dependent on the concentration of the antibiotic treatment. Again, no fungal spores were observed in the slide smear preparations suggesting that nystatin successfully removed contaminating fungi during the treatments. CFU ml 1 (a) (b) (c) Discussion Based on the results from trial 1, AB1 was the least effective treatment, attributable to the lower concentration of antibiotics compared with the other treatments. There was an insignificant difference in bacterial numbers between cultures treated with AB1 compared with the control on both KMV and By+ agar. Either the mode of action of this combination of antibiotics may not be effective against the types of bacteria present, or there may have been antibiotic resistance in these particular thraustochytrid cultures. Bacterial numbers increased on day 6, after treatment with AB3 on both SWC and KMV agars. AB3 did not contain ampicillin, rifampicin or tetracycline. The most effective combination of antibiotics was 0 1 2 Time (Days) 3 4 Figure 3 Bacterial growth recorded on three agar types during antibiotic trial 2. (a) Seawater complete agar, (b) Modified Vishniacs agar, (c) By+ agar. CFU; colony forming unit. Solid line is the control culture, dotted line is treatment AB2, and dashed line is AB2-B. Error bars are equal to one standard deviation. 350 Journal of Applied Microbiology 112, 346 352 ª 2011 The Society for Applied Microbiology

S.L. Wilkens and E.W. Maas Axenic isolation of marine thraustochytrids from NZ AB2 which contained a higher concentration of streptomycin penicillin and the addition of rifampicin. It appears that the combined effect of the higher concentration of streptomycin penicillin combined with rifampicin was able to reduce the bacterial load in the cultures. Results from trial 2 showed no increases in bacterial number on the three growth media used, for each treatment, over the 4 days of the trial suggesting that these bacterial populations are not resistant to the antibiotics being used, and the combination used effectively targets the specific bacterial population found in thraustochytrid isolations from these marine environments. However, the speed at which bacterial reduction was achieved was significantly more rapid after treatment with AB2-B. Antibiotics have been widely used to obtain axenic cell cultures and their mode of action has been well studied. In these trials, six antibiotics were used in an attempt to provide the optimal bacteriocidal effect whilst not harming the thraustochytrid cells. Streptomycin penicillin and ampicillin are both b-lactam antibiotics, broad spectrum and effect cell wall synthesis in both gram positive and negative bacteria. These antibiotics are harmless to microalgae and been used in thraustochytrid culture before (Bongiorni et al. 2005; Perveen et al. 2006; Wong et al. 2008). Rifampicin inhibits DNA-dependent RNA-polymerase within the bacterial cell wall, thus preventing transcription to RNA. Na-Nalidixic acid is effective against both gram positive and negative bacteria by inhibiting growth and reproduction by blocking DNA replication. Both tetracycline and gentamicin inhibit protein synthesis in gram negative bacteria, although tetracycline has also been shown to be effective against gram positive bacteria. Nystatin is a polyene antifungal drug which acts to disrupt fungal cell walls in a broad range of marine fungi. We suggest that the axenic isolation of thraustochytrid colonies can be successfully achieved by two consecutive 24-h antibiotic treatments of 500 mg l )1 streptomycin penicillin, 50 mg l )1 rifampicin and 10 mg l )1 nystatin, followed by serial agar plating onto growth media also containing this combination of antibiotic and antifungal agents. Large-scale culture of thraustochytrids has the potential to be developed as a commercial source of PUFA s and other commercially important products such as antioxidants, pigments, secondary metabolites, polysaccharides and enzymes (Lewis et al. 1999; Raghukumar 2008). Thraustochytrids clearly represent a potentially competitive player in the PUFA market; however, considerable work is required before the production of oil from these organisms significantly increases its share of the market for PUFA-rich products (Lewis et al. 1999). To achieve this aim, a reliable collection and isolation protocol for PUFA-producing strains must be established. Three crucial steps in the development of biotechnological technologies from thraustochytrids include screening, media and culture optimization and development of large-scale fermentation protocols. Here, we present a quick, reliable method to obtain axenic cultures of thraustochytrids isolated from an estuarine environment in northern New Zealand, a preliminary step in the development of biotechnology of thraustochytrids. It is likely that this combination of antibiotics and antifungal will be successful in eliminating microbes from thraustochytrid cultures collected from a range of marine environments as they are broad spectrum and target both gram positive and negative bacterium. 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